US8357836B2 - Agrobacterium-mediated method for producing transformed maize or rice - Google Patents

Agrobacterium-mediated method for producing transformed maize or rice Download PDF

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US8357836B2
US8357836B2 US12/935,525 US93552509A US8357836B2 US 8357836 B2 US8357836 B2 US 8357836B2 US 93552509 A US93552509 A US 93552509A US 8357836 B2 US8357836 B2 US 8357836B2
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agrobacterium
plant
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gene
coculture
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Yuji Ishida
Yukoh Hiei
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Kaneka Corp
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Japan Tobacco Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • C12N15/821—Non-antibiotic resistance markers, e.g. morphogenetic, metabolic markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • C12N15/821—Non-antibiotic resistance markers, e.g. morphogenetic, metabolic markers
    • C12N15/8212—Colour markers, e.g. beta-glucoronidase [GUS], green fluorescent protein [GFP], carotenoid

Definitions

  • the present invention relates to a novel Agrobacterium -mediated method for producing a transformed plant.
  • Agrobacterium -mediated gene transfer is universally used as a transformation method for dicotyledons. Although it has been understood that hosts of Agrobacterium are limited only to dicotyledons and Agrobacterium has no ability to infect monocotyledons (De Cleene and De Ley 1976), some attempts have been made to transform monocotyledons through Agrobacterium -mediated method.
  • Gould et al. injured maize growing points with a needle and then inoculated these growing points with super-virulent Agrobacterium EHA1 carrying the kanamycin resistance gene and the GUS gene, followed by kanamycin selection on the treated growing points to obtain a resistant plant.
  • Agrobacterium EHA1 carrying the kanamycin resistance gene and the GUS gene
  • kanamycin selection on the treated growing points to obtain a resistant plant.
  • Mooney et al. attempted to introduce the kanamycin resistance gene into wheat embryos by using Agrobacterium .
  • the embryos were enzymatically treated to injure their cell walls, and then inoculated with Agrobacterium .
  • the treated calli very few calli were grown that appeared to be resistant to kanamycin, but no whole plant was regenerated from these calli.
  • Southern analysis to confirm the presence of the kanamycin resistance gene, all the resistant calli were found to have a structural mutation in the transgene (Mooney et al. 1991).
  • Raineri et al. performed super-virulent Agrobacterium A281 (pTiBo542) treatment on 8 varieties of rice whose scutellum had been injured, and they confirmed tumorous tissue growth in 2 varieties of Nipponbare, Fujisaka 5. Further, when rice embryos were inoculated with Agrobacterium carrying a Ti plasmid modified to have the kanamycin resistance gene and the GUS gene wherein hormone synthesis genes in T-DNA have been removed, the growth of kanamycin-resistant calli was observed. In these resistant calli, GUS gene expression was observed, but no transformed plant was obtained. Based on these results, Raineri et al. have recognized that the Agrobacterium T-DNA was introduced into rice cells (Raineri et al. 1990).
  • a transformed plant is obtained from a callus derived from an Agrobacterium -inoculated scutellum or immature embryo through the steps of allowing a transformed callus to selectively grow on a medium containing a herbicide component or an antibiotic, and placing the resulting transformed cell clump onto a regeneration medium to induce regeneration (Deji et al., 2000; Negrotto et al., 2000; Nomura et al., 2000a; Nomura et al., 2000b; Taniguchi et al., 2000; Frame et al., 2002; Zhang et al., 2003; Frame et al. 2006).
  • selection of transformed cells is imperative for production of transformed plants, and it has been believed that plant transformation cannot succeed in the absence of this step (Potrykus et al., 1998; Erikson et al., 2005; Joersbo et al., 2001).
  • selection of transformed cells is accomplished as follows: a plant material is introduced with a gene resistant to a drug that inhibits proliferation of non-transformed cells, and then cultured with a medium containing this drug, whereby only transformed cells expressing the drug resistance gene integrated into the plant cell genome are allowed to selectively proliferate.
  • selection marker genes include genes conferring resistance to herbicides or antibiotics (Kuiper et al. 2001). Genes conferring resistance to herbicides include the bar gene and the EPSP gene (De Block et al., 1987; Comai et al., 1985), while genes conferring resistance to antibiotics include the NPTII gene and the HPT gene (Bevan et al., 1983; Waldron et al., 1985), each of which is often used as a selection marker gene for plant transformation.
  • genes conferring the ability to metabolize a specific sugar(s), e.g., the PMI gene and the Xy1A gene are also effective as selection marker genes.
  • selection marker genes based on the mechanism for allowing transformed cells to selectively proliferate, many genes have been reported in addition to those mentioned above.
  • a selection step of allowing transformed cells to selectively proliferate is regarded as essential.
  • transformed seeds are obtained without any selection step (Bent, 2000).
  • a selection step using an antibiotic resistance gene or the like is required.
  • the methods previously used for plant transformation must involve a selection step for isolating transformed cells from non-transformed cells.
  • This selection step requires a selection marker gene used for selection purposes, in addition to a gene of interest (GOI gene), as shown above.
  • GOI gene gene of interest
  • a reaction caused by the action of a protein or enzyme produced upon expression of this selection marker gene e.g., herbicide resistance, antibiotic resistance or fluorescence emission
  • a very few transformed cells among many non-transformed cells can be distinguished and grown to obtain a transformed plant.
  • selection marker gene is no longer required for a produced transformed plant, and many common consumers are insecure about the use of transformed plants because when selection marker genes remain in the transformed plants, the risk of spreading herbicide resistance genes or antibiotic resistance genes to normal non-recombinant plants via transformants is not negligible.
  • many types of selection marker genes are reported, but they have limited use depending on the species of plants, which will cause a problem when multiple genes are introduced separately. Further, although some techniques are reported for removing a selection marker gene from a transformant, these techniques require enormous efforts, including use of a longer culture period than that of standard transformation and/or isolation of selection marker-free plants among progeny plants.
  • Conventional methods for Agrobacterium -mediated gene transfer into monocotyledons must involve a selection step for isolating a transformed cell, tissue, organ or whole plant through introduction of a selection marker gene.
  • the present invention aims to develop and provide a method by which a transformed plant can be obtained without such a selection step.
  • the present invention includes, but is not limited to, the following embodiments as preferred ones.
  • An Agrobacterium -mediated method for producing a transformed plant which comprises:
  • said method comprises transformation enhancement
  • said method does not comprise selection of transformed cells with a selective drug in any step from coculture to regeneration.
  • said method further does not comprise any step for culturing the cocultured tissue with a callus growth medium between the coculture step and the regeneration step.
  • the method according to any one of Embodiments 1 to 3, wherein the transformation enhancement in 1) is intended to improve the efficiency of introducing a gene of interest into plant cells, to improve the efficiency of inducing a callus from an immature embryo, or to improve the efficiency of regeneration from a transformed callus.
  • the present invention provides an Agrobacterium -mediated method for producing a transformed plant.
  • the present invention is directed to a method for Agrobacterium -mediated gene transfer into a plant, which is based on the finding that enhancement of transformation efficiency eliminates the need to introduce a selection marker gene.
  • the method of the present invention is an Agrobacterium -mediated method for producing a transformed plant, which comprises:
  • said method comprises transformation enhancement
  • said method does not comprise selection of transformed cells with a selective drug in any step from coculture to regeneration.
  • transformation enhancement refers to treatment by which the percentage of the resulting transformed plants is increased. Specific examples include, but are not limited to, those intended to improve the efficiency of introducing a gene of interest into plant cells, to improve the efficiency of inducing a callus from an immature embryo, and to improve the efficiency of regeneration from a transformed callus.
  • transformation enhancement includes, but is not limited to, the following or any combination thereof:
  • thermal treatment, centrifugation, thermal treatment and centrifugation, pressurization, and addition of a powder are each intended to improve the efficiency of gene transfer, while addition of silver nitrate, copper sulfate or carbenicillin has the effect of improving the efficiency of callus induction.
  • addition of copper sulfate to the regeneration medium is intended to improve the efficiency of regeneration.
  • thermal treatment may be accomplished, for example, as described in WO98/54961.
  • a plant material is treated at 33° C. to 60° C., preferably at 37° C. to 52° C., for 5 seconds to 24 hours, preferably for 1 minute to 24 hours before being contacted with Agrobacterium.
  • Centrifugation may be accomplished, for example, as described in WO02/12520.
  • a plant material is treated at a centrifugal acceleration of 100 G to 250,000 G, preferably, 500 G to 200,000 G, more preferably 1000 G to 150,000 G, for 1 second to 4 hours, more preferably for 1 second to 2 hours before being contacted with Agrobacterium.
  • Thermal treatment and centrifugation may be accomplished, for example, as described in WO02/12521.
  • Conditions used for thermal treatment and centrifugation may be, for example, the same as those mentioned above.
  • Pressurization may be accomplished, for example, as described in WO2005/017169. Pressurization is performed preferably within the range of, but not limited to, 1.7 to 10 atm, more preferably 2.4 to 8 atm.
  • Addition of silver nitrate and/or copper sulfate to the coculture medium can be found, for example, in Zhao et al. 2001, Ishida et al. 2003, and WO2005/017152.
  • Silver nitrate and/or copper sulfate may be added to the coculture medium at a concentration of 1 to 50 ⁇ M, preferably 1 to 10 ⁇ M.
  • Inoculation with Agrobacterium in the presence of a powder may be accomplished, for example, as described in WO2007/069643. More specifically, an Agrobacterium suspension and a powder may be mixed together and inoculated into a plant material, or alternatively, a plant and a powder may be mixed together and then inoculated with Agrobacterium , by way of example. Any powder may be used for this purpose, including porous powders, glass wool or activated charcoal, preferably porous ceramics, glass wool or activated charcoal, more preferably hydroxyapatite, silica gel or glass wool.
  • N6 inorganic salts to the callus growth medium (Zhao et al. 2001) is accomplished by addition of N6 inorganic salts (Chu 1978) to the callus growth medium.
  • cysteine may be added to the coculture medium at 10 mg/l to 1 g/l, preferably 50 mg/l to 750 mg/l, more preferably 100 mg/l to 500 mg/l.
  • Addition of carbenicillin to the medium in the callus growth and/or regeneration step following the coculture step may be accomplished as described in Zhao et al. 2001 or Ishida et al. 2003.
  • Carbenicillin may be added at a concentration of 50 mg/l to 500 mg/l, preferably 100 mg/l to 300 mg/l to a medium for callus growth and/or during the regeneration step. It should be noted that carbenicillin, which is an antibiotic, has little toxicity to plants and can be used for the purpose of preventing microbial growth in the medium.
  • these treatments are more preferably combined as appropriate.
  • thermal treatment and centrifugation powder treatment, addition of AgNO 3 and/or CuSO 4 to the coculture medium, and addition of carbenicillin as an antibiotic in the callus growth medium and/or in the regeneration medium.
  • thermal treatment and/or centrifugation most preferably in combination with addition of carbenicillin as an antibiotic in the callus growth medium and/or in the regeneration medium.
  • preferred transformation enhancement is thermal treatment, centrifugation, thermal treatment and centrifugation, pressurization, addition of AgNO 3 and/or CuSO 4 to the coculture medium, or inoculation with Agrobacterium in the presence of a powder, addition of carbenicillin as an antibiotic in the callus growth medium and/or in the regeneration medium, or any combination thereof.
  • the inventors of the present invention have succeeded in sufficiently increasing the number of transformed whole plants among finally obtained regenerated whole plants upon these treatments, and have found that a sufficient number of transformed whole plants can be obtained without selection of transformed cells, thereby establishing a practicable method for selection-free transformation.
  • the present invention is characterized in that it does not comprise selection of transformed cells based on the properties of a nucleic acid to be introduced by Agrobacterium in any step from coculture to regeneration required for plant transformation.
  • selection of transformed cells based on the properties of a nucleic acid to be introduced by Agrobacterium .
  • selection of transformed cells with a selective drug and a resistance gene for the selective drug can be presented.
  • Selection of transformed cells with a selective drug and a resistance gene for the selective drug is intended to mean that cells are cultured in a medium supplemented with a drug for selection of a transformed plant to select transformed cells based on the presence or absence of resistance to the selective drug, in any step from coculture to regeneration required for plant transformation.
  • the present invention is completely free from such a step.
  • antibiotics and/or herbicides examples include antibiotics and/or herbicides.
  • Antibiotics conventionally used include those having toxicity to plants, such as hygromycin, kanamycin or blasticidin S, and herbicides conventionally used include phosphinothricin, bialaphos or glyphosate, etc.
  • DNA inserted into T-DNA in Agrobacterium needs to comprise not only a gene to be expressed in a plant, but also a resistance gene for a selective drug.
  • a resistance gene for a selective drug is known in the art.
  • the hygromycin resistance gene should be introduced from Agrobacterium into the plant.
  • a nucleic acid to be introduced by Agrobacterium does not need to carry a resistance gene for a selective drug, i.e., a selection marker gene.
  • a selection marker gene i.e., a selection marker gene.
  • a transformed plant may also be selected based on “auxotrophic selection” (e.g., “sugar requirement”) of plant cells.
  • sugars assimilable by plant cells include sucrose, glucose and so on, but it is known that mannose cannot be assimilated. Thus, when cultured with a medium containing mannose as the only carbon source, plant tissues will die because there is no assimilable sugar. Selection based on sugar requirement relies on this principle.
  • a gene enabling the assimilation of sugars that are generally not assimilable by plant cells should be introduced from Agrobacterium into the plant tissue.
  • a gene is known in the art and, for example, the PMI gene, the xylose isomerase gene or the like may be used for this purpose.
  • a nucleic acid to be introduced does not need to comprise such a gene.
  • an easily detectable gene may be introduced as a screening indicator to select a transformed plant based on the presence or absence of expression of this gene.
  • a gene serving as a screening indicator include the GFP gene, etc.
  • a nucleic acid to be introduced does not need to comprise such a gene.
  • selection marker gene is intended to mean an additional gene that is introduced into a plant together with a gene of interest (GOI gene) to be transformed, for the purpose of selecting transformed cells from among many non-transformed cells.
  • GOI gene gene of interest
  • examples of such a selection marker gene include, but are not limited to, herbicide resistance genes, antibiotic resistance genes, fluorescent genes and so on.
  • An Agrobacterium -mediated method for producing a transformed plant generally involves all or part of Steps I to V shown below.
  • Plants targeted by the present invention are those to which Agrobacterium -mediated transformation is applicable. Preferred are monocotyledons. Monocotyledons used in the method of the present invention are preferably Gramineae plants including, but not limited to, rice, maize, barley, wheat, sorghum and so on. The most preferred plant used in the method of the present invention is rice or maize.
  • the term “plant material” is intended to encompass all embodiments of such a plant to be used for Agrobacterium -mediated plant transformation, including a cell, a leaf, a root, a stem, a bud, a flower (including a stamen, a pistil, etc.), a fruit, a seed, a germinated seed or a plant tissue of any other site, a growing point, an explant, an immature embryo, a callus or an embryoid-like tissue (hereinafter collectively referred to as “callus or the like” or simply “callus”), or a perfect whole plant.
  • callus or the like an embryoid-like tissue
  • a preferred form of the plant material used in the method of the present invention is an immature embryo or a callus, most preferably an immature embryo.
  • plant cell plant tissue
  • perfect whole plant used herein have the same meanings as commonly used in the art.
  • the term “immature embryo” is intended to mean the embryo and scutellum of an immature seed under maturation after pollination.
  • the stage (maturation phase) of the immature embryo used in the method of the present invention is not limited in any way, and it may be collected at any stage after pollination. It is most preferably at a post-pollination stage of 2 days or more.
  • an immature embryo capable of inducing a callus having the ability to grow and regenerate a normal whole plant by the method described later after the transformation described later.
  • an immature embryo is preferably an immature embryo of an inbred line, F1 between inbred lines, F1 between an inbred line and an open-pollinated variety, or a commercially available F1 variety.
  • the term “callus” is intended to mean an undifferentiated cell clump under uncontrolled growth.
  • differentiated cells of a plant tissue may be cultured in a medium containing a plant growth regulator such as auxin (e.g., 2,4-D) or cytokinin (referred to as a dedifferentiation medium).
  • a plant growth regulator such as auxin (e.g., 2,4-D) or cytokinin (referred to as a dedifferentiation medium).
  • This treatment for obtaining a callus is referred to as dedifferentiation treatment, and this process is referred to as dedifferentiation process.
  • a plant tissue, an immature embryo or the like is excised from a whole plant, a seed or the like to prepare a plant material preferred for use in transformation.
  • Preparation of a plant material may be accomplished in any known manner. If desired, a plant material may be cultured before being infected with Agrobacterium.
  • the plant material used in the present invention is inoculated with Agrobacterium .
  • the term “inoculation” or “inoculated” used herein is intended to mean that Agrobacterium is contacted with a plant material, and various techniques for Agrobacterium inoculation are known in the art. Examples of such techniques include those in which a plant material is added to a suspension of Agrobacterium suspended in a liquid medium, those in which an Agrobacterium suspension is directly added dropwise to a plant material on a coculture medium, those in which an Agrobacterium suspension is injected into a plant material, and those in which a plant material is immersed in an Agrobacterium suspension and incubated under reduced pressure.
  • the Agrobacterium -inoculated plant material used in the present invention is not limited to those inoculated with Agrobacterium by these techniques.
  • various additives e.g., acetosyringone, surfactants, porous ceramics
  • acetosyringone e.g., acetosyringone, surfactants, porous ceramics
  • Agrobacterium that can be used in the present invention may be any known species of Agrobacterium , preferably Agrobacterium tumefaciens or Agrobacterium rhizogenes .
  • examples of Agrobacterium include, but are not limited to, LBA4404, EHA101 and AGL1, C58C1 and others.
  • a soil bacterium Agrobacterium ( Agrobacterium tumefaciens ) has been known to cause crown gall disease in many dicotyledons since a long time ago, and in 1970s, it was found that its Ti plasmid was involved in pathogenicity, and that T-DNA, a part of the Ti plasmid, was integrated into the plant genome. Subsequently, it has been shown that T-DNA contains genes involved in synthesis of hormones (cytokinin and auxin) required to induce cancer, and these genes are expressed in plants although they are bacterial genes.
  • hormones cytokinin and auxin
  • a group of genes located in the virulence region (vir region) on the Ti plasmid are required for excision of T-DNA and its transfer to plants, and border sequences flanking T-DNA are required for excision of T-DNA.
  • Agrobacterium rhizogenes another species of Agrobacterium , has a similar system based on Ri plasmid (e.g., FIGS. 3 and 4 of JP 2000-342256 A).
  • T-DNA Since T-DNA is integrated into the plant genome upon Agrobacterium infection, it was expected that a desired gene would also be integrated into the plant genome when inserted onto T-DNA. However, it was difficult to insert a gene onto T-DNA on Ti plasmid by standard genetic engineering techniques, because Ti plasmid is as large as 190 kb or more. For this reason, there was developed a method for inserting a foreign gene onto T-DNA.
  • disarmed strains in which hormone synthesis genes have been removed from T-DNA of tumor-inducing Ti plasmid were prepared, including LBA4404 (see Hoekema, A., et al., (1983), Nature, Vol. 303, p. 179-180), C58C1 (pGV3850), GV3Ti11SE, etc.
  • LBA4404 see Hoekema, A., et al., (1983), Nature, Vol. 303, p. 179-180
  • C58C1 pGV3850
  • GV3Ti11SE GV3Ti11SE
  • the second method is called the binary vector method, which is based on the finding that the vir region is required for integration of T-DNA into plants but needs not be located on the same plasmid to exert its functions.
  • This vir region contains virA, virB, virC, virD, virE and virG (Dictionary of Plant Biotechnology, published by Enterprise (1989)), and the vir region is intended to mean a region containing all of these virA, virB, virC, virD, virE and virG.
  • the term “binary vector” refers to a vector carrying T-DNA integrated into a small plasmid replicable in both Agrobacterium and E. coli , and this binary vector is introduced into Agrobacterium having a disarmed Ti plasmid before use.
  • a binary vector into Agrobacterium can be accomplished in any known manner, for example, by electroporation or triparental mating.
  • Examples of a binary vector include pBIN19, pBI121, pGA482, etc., and many new binary vectors have been constructed based on these vectors and used for transformation.
  • Similar vectors have been constructed and used for transformation.
  • Agrobacterium A281 is a super-virulent strain that has a wide host range and higher transformation efficiency than other strains. This characteristic is attributed to pTiBo542, i.e., the Ti plasmid of A281.
  • pTiBo542 i.e., the Ti plasmid of A281.
  • Two novel systems have been developed so far with the use of pTiBo542.
  • One of them uses strains EHA101 and EHA105, each carrying a disarmed Ti plasmid of pTiBo542, and is used for transformation of various plants as a system with high transforming ability by applying these strains to the binary vector system described above.
  • This system is a kind of binary vector system because it consists of a disarmed Ti plasmid having the vir region (virA, virB, virC, virD, virE and virG (each hereinafter also referred to as a “vir fragment region”)) and a plasmid having T-DNA.
  • vir fragment region a disarmed Ti plasmid having the vir region
  • virG virD, virE and virG
  • T-DNA plasmid having T-DNA
  • a super-binary vector carrying a fragment of the vir region substantially free from at least one of the vir fragment regions (preferably a fragment including at least virB or virG, more preferably a fragment including virB and virG) in the plasmid having T-DNA, i.e., a binary vector.
  • homologous recombination via triparental mating can be used as a convenient technique to introduce the T-DNA region carrying a desired gene into Agro
  • any species of Agrobacterium may be used as a host, and preferred for use is Agrobacterium tumefaciens (e.g., Agrobacterium tumefaciens LBA4404 (see Hoekema, A., et al., (1983), Nature, Vol. 303, p. 179-180) and EHA101 as mentioned above).
  • Agrobacterium tumefaciens e.g., Agrobacterium tumefaciens LBA4404 (see Hoekema, A., et al., (1983), Nature, Vol. 303, p. 179-180) and EHA101 as mentioned above).
  • the benefit of the present invention can be obtained by using any gene transfer system as long as it relies on the expression of the genes in the virulence (vir) region in Agrobacterium.
  • the benefit of the present invention can be obtained by using any vector system, including intermediate vectors, binary vectors, super-virulent binary vectors and super-binary vectors as described above, with super-virulent binary vectors and super-binary vectors being preferred because of improved transformation efficiency.
  • super-binary vectors are preferred for use when a target plant is maize.
  • the same benefit can also be obtained by using different vector systems modified from the above vectors (e.g., by excising a part or all of the vir region in Agrobacterium and additionally integrating it into a plasmid, or by excising a part or all of the vir region and introducing it into Agrobacterium as a part of a new plasmid).
  • a desired gene to be introduced into plants can be inserted in a standard manner into a restriction enzyme site in the T-DNA region of the above plasmid.
  • desired DNA may not be easily introduced into the T-DNA region by standard subcloning techniques.
  • desired DNA can be introduced by homologous recombination in cells of Agrobacterium via triparental mating.
  • the size of a gene to be introduced is preferably, but not limited to, about 100 bp to 200 kbp.
  • Agrobacterium e.g., Agrobacterium tumefaciens
  • introduction of a plasmid into Agrobacterium can be accomplished in a conventional manner, for example, by triparental mating as described above, electroporation, electroinjection, chemical treatment with PEG, etc.
  • a gene to be introduced into plants is basically located between the border sequences flanking T-DNA, as in the case of conventional techniques. However, only one border sequence may exist because the plasmid is circular, or alternatively, three or more border sequences may exist when multiple genes are to be located at different sites.
  • the gene may also be located on the Ti or Ri plasmid or on another plasmid in Agrobacterium . Alternatively, it may also be located on multiple types of plasmids.
  • Inoculation of a plant material with Agrobacterium may be accomplished, for example, by simply establishing contact between the plant material and Agrobacterium . Inoculation may be accomplished by either standard inoculation or drop inoculation.
  • Standard inoculation is a technique in which a plant material is mixed with an Agrobacterium suspension (inoculum) to ensure immersion of the plant material in the suspension, and the immersed plant material is collected and placed onto a medium to effect coculture for inoculation.
  • Agrobacterium suspension inoculum
  • it may be accomplished by preparing an Agrobacterium suspension at a cell density of about 10 6 to 10 11 cfu/ml, immersing a plant material in this suspension for about 3 to 10 minutes, and then coculturing the plant material on a solid medium for several days.
  • Drop inoculation is a technique in which an Agrobacterium suspension is added dropwise onto a plant material placed on a medium, and after the suspension added dropwise is dried, the plant material is transferred to another site on the same medium or placed onto another medium to effect coculture for inoculation.
  • plant cells inoculated with Agrobacterium as described above are cultured together with the Agrobacterium with a medium containing an auxin member to thereby ensure DNA introduction from the Agrobacterium into the plant cells.
  • a plant material is cocultured with Agrobacterium during or after infection of the plant material with Agrobacterium and before removal of Agrobacterium.
  • coculture medium The medium used in this step is referred to herein as “coculture medium.”
  • any known medium may be used, including LS-AS medium, nN6-AS medium, as well as N6S3-AS medium, 2N6-AS medium (see Hiei, Y., et al., (1994), The Plant Journal, Vol. 6, p. 271-282) and so on.
  • the coculture medium is preferably supplemented with an auxin member(s). Since auxin members generally have the ability to induce dedifferentiation in plant materials, almost every plant material is partially or fully turned into a dedifferentiated tissue (callus) during this step.
  • auxin members include 3,6-dichloro-o-anisic acid (dicamba), 4-amino-3,5,6-trichloropicolinic acid (picloram), 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and/or triiodobenzoic acid (TIBA).
  • the coculture medium is free from any auxin member other than dicamba, picloram, 2,4-D and 2,4,5-T.
  • the total amount of auxin members (e.g., dicamba, picloram, 2,4-D, 2,4,5-T) in the coculture medium is preferably, but not limited to, 0.1 to 5.0 mg/l, more preferably 0.5 to 3.0 mg/l, even more preferably 1.0 to 2.0 mg/l, and most preferably 1.5 mg/l.
  • transformation efficiency is further improved enough to produce a transformed plant without the need to introduce a selection marker gene, particularly when a substance with auxin activity belonging to benzoic herbicides among auxin members, is added to the coculture medium.
  • benzoic herbicides are classified into (i) benzoic acid type, (ii) salicylic acid type, (iii) picolinic acid type, and (iv) terephthalic acid type (Takematsu, 1982).
  • herbicides of (iv) terephthalic acid type have no auxin activity, and hence preferred herbicides are of (i) benzoic acid type, (ii) salicylic acid type or (iii) picolinic acid type, more preferably of either (ii) salicylic acid type or (iii) picolinic acid type.
  • the coculture medium is most preferably supplemented with a substance with auxin activity belonging to benzoic herbicides.
  • the coculture medium is preferably supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D).
  • the term “culture” in this step is intended to mean that a plant material is placed on a solidified coculture medium or in a liquid coculture medium and is allowed to grow at an appropriate temperature under appropriate light/dark conditions for an appropriate period.
  • the coculture medium may be solidified by addition of any solidifying agent known in the art, including agarose.
  • the culture temperature in this step may be selected as appropriate, and is preferably 20° C. to 35° C., more preferably 25° C.
  • culture in this step is preferably accomplished in the dark, but is not limited thereto.
  • the culture period in this step may also be selected as appropriate, and is preferably 1 to 10 days, more preferably 7 days.
  • a callus growth step has generally been regarded as a necessary step.
  • callus growth medium refers to a medium containing plant hormones and nutrients suitable for division and proliferation of cells in a dedifferentiated state. In standard transformation tests, it is also used as a “selective medium” by being supplemented with a drug (selection pressure), which inhibits the proliferation of non-transformed cells, to thereby ensure selective proliferation of transformed cells.
  • selection pressure selection pressure
  • the plant material after the above coculture step is cultured with a medium containing an auxin member to select a transformant based on the presence or absence of gene transfer.
  • the medium used in this step is referred to herein as “selective medium” and contains a selective drug or the like for selection based on the presence or absence of gene transfer.
  • This step is repeated for several rounds in the conventional methods while varying the composition of medium components.
  • the selective drug concentration may be elevated at each round to ensure a higher reliability of drug selection, so that the possibility of obtaining a transformed whole plant can be increased.
  • This selection step is preferably repeated for at least 2 rounds, more preferably 3 rounds. When repeated for several rounds, this step requires a period of about 10 days to 3 weeks for each round, and the total period required for several rounds of selection is about 5 to 10 weeks. Thus, this step is the most time-consuming step in Agrobacterium -mediated plant transformation.
  • this step has been regarded as an essential step.
  • transformation enhancement ensures successful transformation without the need to select transformed cells with a selective drug in any step from coculture to regeneration, including during the callus growth step, and thus have arrived at the present invention.
  • this step can preferably be eliminated.
  • the present invention does not comprise any step for culturing the cocultured tissue with a callus growth medium between the coculture step and the regeneration step. This allows a further improvement in manipulation efficiency and time saving in the transformation process, and hence the inventors have found that a transformed plant can be obtained more efficiently.
  • Examples 1-4 and 6-7 described herein later demonstrate successful cases of plant transformation without requiring any callus growth step.
  • the callus growth step may be performed without adding a selective drug to the callus growth medium. In this case, callus growth will occur but “selection” of transformants will not be performed.
  • This step involves regeneration of the resulting cell clump and growth of the regenerated plant to obtain a perfect whole plant, if desired.
  • Regeneration of a perfect whole plant from the resulting transformed cells may be accomplished in any known manner (e.g., Hiei, Y., et al., (1994), The Plant Journal, Vol. 6, p. 271-282; and Ishida, Y., et al., (1996), Nature Biotechnology, Vol. 14, p. 745-750).
  • This step is essential both in conventional methods and in the present invention. It has been believed that selection of transformants with a selective drug is also essential in the regeneration step.
  • the plant material after the coculture step is cultured with a regeneration medium containing a selective drug and then tested for the presence or absence of resistance to the selective drug.
  • the present invention is characterized in that it does not comprise selection of transformed cells with a selective drug in any step from coculture to regeneration required for plant transformation. Thus, the present invention does not comprise selection with a selective drug even in the regeneration step.
  • the medium used in this step is referred to herein as “regeneration medium,” which is characterized by containing no auxin member.
  • regeneration medium examples include those based on LS inorganic salts or N6 inorganic salts, more specifically LSZ medium.
  • the “regeneration medium” is free from any selective drug.
  • regeneration used herein is intended to mean that a fully or partially dedifferentiated plant material acquires again the properties of the original plant material or whole plant.
  • a dedifferentiated tissue When subjected to the regeneration step, a dedifferentiated tissue will be able to regenerate, whereby a perfect transformed whole plant can be obtained. Determination of whether regeneration has occurred or not may be readily accomplished by observation of plant morphology, for example, by determining whether a specific differentiated plant organ (e.g., stem, leaf) develops from a dedifferentiated tissue.
  • a specific differentiated plant organ e.g., stem, leaf
  • vigor is intended to mean the growth vigor of a regenerated plant.
  • the method of evaluation of vigor is not limited to this technique, and appropriate modifications may be made to well-known techniques, e.g., depending on the type of target to be evaluated.
  • culture in this step is intended to mean that a plant tissue is placed on a solidified regeneration medium or in a liquid regeneration medium and is allowed to grow at an appropriate temperature under appropriate light/dark conditions for an appropriate period.
  • the regeneration medium may be solidified, for example, with agar or the like as shown above.
  • the culture temperature in this step may be selected as appropriate, and is preferably 20° C. to 35° C., more preferably 25° C.
  • culture in this step is preferably accomplished in the light for 16 to 24 hours a day, but is not limited thereto.
  • the culture period in this step may also be selected as appropriate, and is preferably 7 to 21 days, more preferably 14 days.
  • a perfect transformed whole plant can be easily obtained in a manner known in the art.
  • the presence or absence of the transgene is confirmed for each of the resulting regenerated plants to specify a transformed whole plant.
  • PCR or Southern analysis may be preferably used for this purpose.
  • isolation may be easily accomplished by confirming the phenotype of the transgene.
  • the present invention ensures stable and efficient transformation in a desired plant without the need to introduce a plant selection marker gene.
  • FIG. 1 shows the results of Southern blot analysis performed on regenerated whole plants of the cultivar Yukihikari obtained by selection-free transformation.
  • FIG. 2 shows the results of Southern blot analysis performed on regenerated whole plants of the variety A188 obtained by selection-free transformation.
  • FIG. 3 shows the results of Southern blot analysis performed on regenerated whole plants (T0) of the variety A188 obtained by selection-free transformation and their T1 progeny.
  • LS-inf liquid medium containing 100 ⁇ M acetosyringone Agrobacterium strain LBA4404 (pSB134) (Hiei and Komari, 2006) was suspended at about 1.0 ⁇ 10 9 cfu/ml to prepare an inoculum.
  • the thermally-treated and centrifuged immature embryos were mixed with the inoculum, vortexed for 30 seconds, and then allowed to stand for 5 minutes at room temperature.
  • the Agrobacterium -inoculated immature embryos were placed, with their scutella facing up, onto a coculture medium containing dicamba (3,6-dichloro-o-anisic acid) at a concentration of 1.5 mg/l in LS-AS medium (Ishida et al., 1996; solidified with 8 g/l agarose) which had been prepared to exclude 2,4-D (2,4-dichlorophenoxyacetic acid) and contain 5 ⁇ M AgNO 3 and 5 ⁇ M CuSO 4 .
  • the immature embryos cultured in the dark at 25° C. for 7 days were placed onto LSZ medium (Ishida et al., 1996) containing 10 ⁇ M CuSO 4 , and then cultured in the light at 25° C. for about 2 weeks. Leaves of the regenerated whole plants were partially excised and immersed in 0.1 M phosphate buffer (pH 6.8) containing 0.1% Triton X-100 and then allowed to stand at 37° C. for 1 hour. The phosphate buffer was removed and replaced by another phosphate buffer containing 1.0 mM 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronic acid (X-gluc) and 20% methanol. After treatment at 37° C. for 24 hours, GUS gene expression was analyzed.
  • Example 1 The conditions used in Example 1 are summarized below.
  • Transformation enhancement thermal treatment and centrifugation, addition of Ag + and Cu 2+ ions to the medium
  • the embryos were directly subjected to the regeneration step without passing through the callus growth step.
  • pSB134 a super-binary vector carrying the GUS gene and the hygromycin resistance gene (selection marker gene) and also carrying a part of the virulence gene from a super-virulent strain
  • Leaf pieces derived from two immature embryos were all GUS-negative. In leaf pieces collected from the remaining 14 immature embryos, at least one leaf piece showed GUS gene expression in each case. Leaf pieces showing a dotted pattern of expression were observed for 5 immature embryos. Leaf pieces expressing the GUS gene in a striped pattern were observed for 6 immature embryos. There were two immature embryos giving not only leaf pieces showing a dotted pattern of expression but also leaf pieces showing a striped pattern of expression. In this case, a plant showing a striped pattern of expression is predicted to be a chimera between transformed and non-transformed cells, while a plant showing a dotted pattern of expression is predicted to have undergone gene silencing in some transformed cells. GUS-positive leaf pieces whose cut ends were uniformly stained blue were obtained from one immature embryo.
  • Example 2 An inoculum (1 ml) of Agrobacterium strain LBA4404 (pSB134) prepared in the same manner as shown in Example 1 was supplemented with about 80 mg of hydroxyapatite (Bio-Rad). Immature embryos were subjected to pretreatment (thermal treatment at 46° C. for 3 minutes and centrifugation at 20,000 ⁇ g for 10 minutes) to increase gene transfer efficiency for transformation enhancement.
  • the immature embryos (variety: A188) were placed, with their scutella facing up, onto a coculture medium containing dicamba at a concentration of 1.5 mg/l in LS-AS medium (Ishida et al., 1996; solidified with 8 g/l agarose) which had been prepared to exclude 2,4-D and contain 5 ⁇ M AgNO 3 and 5 ⁇ M CuSO 4 .
  • Example 2 The conditions used in Example 2 are summarized below.
  • Transformation enhancement powder treatment, thermal treatment and centrifugation, addition of Ag + and Cu 2+ ions to the medium
  • the embryos were directly subjected to the regeneration step without passing through the callus growth step.
  • pSB134 a super-binary vector carrying the GUS gene and the hygromycin resistance gene (selection marker gene) and also carrying a part of the virulence gene from a super-virulent strain
  • LS-inf liquid medium containing 100 ⁇ M acetosyringone Agrobacterium strain LBA4404 (pSB124) ( Komari et al., 1996) was suspended to prepare an inoculum.
  • the thermally-treated immature embryos were mixed with the inoculum, vortexed for 30 seconds, and then allowed to stand for 5 minutes at room temperature.
  • the Agrobacterium -inoculated immature embryos (24 in total) were placed, with their scutella facing up, onto a coculture medium containing dicamba (3,6-dichloro-o-anisic acid) or picloram (4-amino-3,5,6-trichloropicolinic acid) at a concentration of 6.8 ⁇ M in LS-AS medium (Ishida et al., 1996; solidified with 8 g/l agarose) which had been prepared to exclude 2,4-D (2,4-dichlorophenoxyacetic acid) and contain 5 ⁇ M AgNO 3 and 5 ⁇ M CuSO 4 .
  • LS-AS medium containing 5 ⁇ M AgNO 3 and 5 ⁇ M CuSO 4 was supplemented with 1.5 mg/l (6.8 ⁇ M) 2,4-D as an auxin member and also used for testing. 24 immature embryos were placed onto this medium.
  • the immature embryos cultured in the dark at 25° C. for 7 days were placed onto LSZ medium (Ishida et al., 1996) containing 10 ⁇ M CuSO 4 , and then cultured in the light at 25° C. for about 2 weeks. Leaves of the regenerated whole plants were partially excised and immersed in 0.1 M phosphate buffer (pH 6.8) containing 0.1% Triton X-100 and then allowed to stand at 37° C. for 1 hour. The phosphate buffer was removed and replaced by another phosphate buffer containing 1.0 mM 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronic acid (X-gluc) and 20% methanol. After treatment at 37° C. for 24 hours, GUS gene expression was analyzed.
  • Example 3 The conditions used in Example 3 are summarized below.
  • Transformation enhancement thermal treatment, addition of Ag + and Cu 2+ ions to the medium
  • the embryos were directly subjected to the regeneration step without passing through the callus growth step.
  • pSB124 a super-binary vector carrying the GUS gene but no selection marker gene and also carrying a part of the virulence gene from a super-virulent strain
  • the immature embryos cocultured with the medium containing particularly dicamba or picloram were found to regenerate into whole plants with higher efficiency, and also resulted in an increased percentage of regenerated plants showing expression of the transgene.
  • the immature embryos cocultured with the 2,4-D-containing medium also showed expression of the transgene, which was slightly lower.
  • a substance belonging to benzoic herbicides is more suitable than a substance belonging to phenoxy herbicides when used as an auxin member to be incorporated into a medium for coculture of Agrobacterium -inoculated immature embryos.
  • the thermally- and centrifugally-pretreated immature embryos and the non-pretreated control immature embryos were each mixed with the inoculum, vortexed for 30 seconds, and then allowed to stand for 5 minutes at room temperature.
  • the Agrobacterium -inoculated immature embryos were placed, with their scutella facing up, onto a coculture medium containing dicamba (3,6-dichloro-o-anisic acid) at a concentration of 6.8 ⁇ M in LS-AS medium (Ishida et al., 1996; solidified with 8 g/l agarose) which had been prepared to exclude 2,4-D (2,4-dichlorophenoxyacetic acid) and contain 5 ⁇ M AgNO 3 and 5 ⁇ M CuSO 4 .
  • the pretreated and non-pretreated immature embryos were provided for testing (75 embryos each). The test was performed in duplicate.
  • the immature embryos cultured in the dark at 25° C. for 7 days were placed onto LSZ medium (Ishida et al., 1996) containing 10 ⁇ M CuSO 4 , and then cultured in the light at 25° C. for about 2 weeks. Leaves of the regenerated whole plants were partially excised and immersed in 0.1 M phosphate buffer (pH 6.8) containing 0.1% Triton X-100 and then allowed to stand at 37° C. for 1 hour. The phosphate buffer was removed and replaced by another phosphate buffer containing 1.0 mM 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronic acid (X-gluc) and 20% methanol. After treatment at 37° C. for 24 hours, GUS gene expression was analyzed.
  • Example 4 The conditions used in Example 4 are summarized below.
  • the embryos were directly subjected to the regeneration step without passing through the callus growth step.
  • pSB124 a super-binary vector carrying the GUS gene but no selection marker gene and also carrying a part of the virulence gene from a super-virulent strain
  • the thermally-treated and centrifuged 75 immature embryos were found to regenerate into 296 shoots. Among them, 5 shoots showed GUS gene expression throughout the leaf tissue. In contrast, the non-pretreated 75 immature embryos were found to regenerate into 291 shoots, although there was no shoot showing GUS gene expression throughout the leaf tissue. In the second test, the thermally-treated and centrifuged immature embryos were found to regenerate into 243 shoots, and there were four GUS-positive shoots showing GUS gene expression throughout the leaf tissue.
  • AA-inf liquid medium (Hiei and Komari, 2006) containing 100 ⁇ M acetosyringone, Agrobacterium LBA4404 (pSB 134) (Hiei and Komari, 2006) was suspended at a concentration of about 1.0 ⁇ 10 9 cfu/ml to prepare an inoculum.
  • the centrifuged immature embryos were placed, with their scutella facing up, onto nN6-As medium (Hiei et al., 2006).
  • the inoculum was added dropwise in 5 ⁇ l volumes, followed by coculture in the dark at 25° C. for 7 days.
  • each immature embryo was divided into 4 or 5 parts with a surgical knife.
  • the divided immature embryos were placed, with their scutella facing up, onto nN6 medium (Hiei et al., 2006) containing 250 mg/l cefotaxime and 100 mg/l carbenicillin, and then cultured in the light at 30° C. for about 10 days.
  • Sections expanded primarily by proliferation of scutellum cells were each further divided into 4 or 5 parts. At this stage, 18 to 25 sections were obtained for each immature embryo.
  • the sections were placed, with their scutella facing up, onto nN6 medium (Hiei et al., 2006) containing 250 mg/l cefotaxime and 100 mg/l carbenicillin, and then cultured in the light at 30° C. for about 2 weeks.
  • nN6 medium Hiei et al., 2006
  • the shoot clump regenerated from each callus was transferred to N6F rooting medium (Hiei et al., 2006) and cultured in the light at 30° C. for about 10 days to obtain a perfect regenerated whole plant. It should be noted that the above medium is free from any selective drug, such as hygromycin, bialaphos or the like.
  • Southern blot analysis was performed in the following manner. From leaves of the regenerated whole plants, DNAs were extracted according to the method of Komari (1989) and digested in an amount of 7 ⁇ g per plant with a restriction enzyme HindIII. The digested DNAs were subjected to 0.7% agarose gel electrophoresis (1.5 V/cm, 15 hours). Southern hybridization was performed according to the method of Sambrook et al. (1989). It should be noted that the probe used was a SaII-SacI (1.9 kb) fragment of pGL2-IG (Hiei et al., 1994), which is a GUS gene fragment.
  • Example 5 The conditions used in Example 5 are summarized below.
  • Transformation enhancement centrifugation, addition of cefotaxime and carbenicillin to the medium
  • the embryos were subjected to the regeneration step through the callus growth step.
  • pSB134 a super-binary vector carrying the GUS gene and the hygromycin resistance gene (selection marker gene) and also carrying a part of the virulence gene from a super-virulent strain
  • Test 1 The test was performed in duplicate (Tests 1 and 2). The results of Test 1 are shown in Table 1.
  • 100 divided sections were obtained from 5 immature embryos, and 100 calli from these sections were placed onto the regeneration medium to obtain 92 whole plants. Among them, 73 whole plants were each analyzed for GUS gene expression in one leaf, indicating that 9 whole plants were GUS-positive transformants showing uniform GUS expression throughout the leaf tissue. The transformation efficiency was 12.3% per regenerated whole plant.
  • Test 2 The results of Test 2 are shown in Table 2.
  • 107 divided sections were obtained from 5 immature embryos, and 107 calli from these sections were placed onto the regeneration medium to obtain 100 whole plants.
  • 95 whole plants were each analyzed for GUS gene expression in one leaf, indicating that 16 whole plants were transformants showing uniform GUS expression.
  • the transformation efficiency in this case was 16.8% per regenerated whole plant.
  • FIG. 1 The results of Southern blot analysis are shown in FIG. 1 .
  • the regenerated whole plants positive for GUS gene expression were confirmed to have the introduced GUS gene in all of the 11 cases tested ( FIG. 1 ).
  • the regenerated whole plants showing GUS gene expression in a dotted pattern were also confirmed to have the GUS gene in all of the 6 cases tested, but the introduced gene in these whole plants tended to be present in a high copy number ( FIG. 1 ).
  • the regenerated whole plants negative for GUS gene expression were also subjected to Southern blot analysis, but there was no band hybridizing to the GUS probe in each of the 7 whole plants tested.
  • scutellum cells in the gene-transferred immature embryos were allowed to proliferate, and calli were randomly extracted therefrom to obtain regenerated whole plants. 10% or more of these regenerated whole plants were found to be transformed whole plants. This indicates that 10% or more of scutellum cells in immature embryos have been transformed by Agrobacterium infection.
  • the transformed maize plants obtained in Examples 3 and 4 were cultivated in a greenhouse. From leaves of these plants, DNAs were extracted according to the method of Komari et al. (Komari et al. (1989)) and digested in an amount of 10 ⁇ g per plant with a restriction enzyme BamHI. The digested DNAs were subjected to 0.7% agarose gel electrophoresis (1.5 V/cm, 15 hours). Southern hybridization was performed according to the method of Sambrook et al. (Sambrook, J., et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). It should be noted that the probe used was a SalI-SacI (1.9 kb) fragment of pGL2-IG (Hiei et al., 1994), which is a GUS gene fragment.
  • Each transformant showed a band(s) hybridizing to the GUS probe.
  • the band pattern differed from transformant to transformant, thus indicating that the transgene was randomly inserted onto the plant chromosome.
  • the transformed maize plants (variety: A188) obtained in Examples 3 and 4 were cultivated in a greenhouse. Pollen was collected from non-transformed A188 and crossed with the floss of each transformed plant to obtain progeny seeds (T1 generation).
  • the resulting seeds were seeded in plastic pots containing potting soil. Leaves were excised from young seedlings at 11 days after seeding, immersed in 0.1 M phosphate buffer (pH 6.8) containing 0.1% Triton X-100, and then allowed to stand at 37° C. for 1 hour. The phosphate buffer was removed and replaced by another phosphate buffer containing 1.0 mM 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronic acid (X-gluc) and 20% methanol. After treatment at 37° C. for 24 hours, GUS gene expression was analyzed.
  • DNAs were extracted according to the method of Komari (1989) and digested in an amount of 10 ⁇ g per plant with a restriction enzyme BamHI. The digested DNAs were subjected to 0.7% agarose gel electrophoresis (1.5 V/cm, 15 hours). Southern hybridization was performed according to the method of Sambrook et al. (1989). It should be noted that the probe used was a SalI-SacI (1.9 kb) fragment of pGL2-IG (Hiei et al., 1994), which is a GUS gene fragment.
  • Table 3 shows the results analyzed for segregation of the GUS gene in progeny plants of transformed maize plants obtained by selection-free transformation.
  • DNA extracted from the transformed plant (T0) of line No. 195 showed a single hybridizing band.
  • T1 progeny plants of this line DNAs extracted from five GUS-positive plants each showed a single band of the same size as in the T0 plant. In contrast, no hybridizing band was detected in DNAs extracted from GUS-negative plants.
  • DNA extracted from the transformed plant (T0) of line No. 169 showed five hybridizing bands.
  • T1 progeny plants of this line DNAs extracted from five GUS-positive plants each showed bands of the same size as in the T0 plant although the number of bands varied from 1, 4 or 5. Since GUS gene expression in T1 plants of line No. 169 showed two-factor segregation, it was predicted that 4 copies and 1 copy of the GUS gene would be located on different chromosomes, respectively. No hybridizing band was detected in DNAs extracted from GUS-negative plants ( FIG. 3 ).

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ES2945315T3 (es) 2015-01-27 2023-06-30 Inst Genetics & Developmental Biology Cas Método para realizar modificaciones específicas de sitio en la planta completa a través de la expresión transitoria de genes
UA125246C2 (uk) 2015-03-16 2022-02-09 Інстітьют Оф Генетікс Енд Дівелопментл Байолоджі, Чайніз Екадемі Оф Сайнсис Спосіб здійснення сайт-спрямованої модифікації рослинних геномів із застосуванням неуспадковуваних матеріалів
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